Following nerve injury, pro-inflammatory cytokines increase within dorsal root ganglia (DRG) and spinal cord where they activate mitogen-activated protein kinases (MAPK), including p38 kinase (p38). Activation of p38 alters multiple proteins in primary afferent fibers and dorsal horn cells, and its inhibition reduces pain behavior following nerve injury. This application proposes to dissect the molecular pathway upstream and downstream of p38 activation in response to spinal nerve ligation, a model of neuropathic pain. Aim 1 will concentrate on nerve injury-induced changes in p38 and its isoforms. Aim 2 will look at upstream regulators and aim 3 at potential downstream effectors. We will systematically examine co-variance of pain behavior (mechanical and thermal sensitivity) with changes in activation and/or expression of cytokines (TNF and IL- 1), MKK3, MKK6, p38 isoforms and MAPKAP-2, a p38 substrate kinase. Changes in protein expression (Western blots) will be followed, in some instances, by a kinase assays to verify activity, and immunohistochemistry to ascertain cellular localization. Spinal cord and the DRG will be examined separately to determine if the cascade differs between the two locations. Inhibition or loss of individual elements in the cascade will allow us to determine what elements "downstream" of the blockade are reduced. Blockade of individual elements will occur using antisense for individual p38 isoforms, knock out mice and receptor specific pharmacological antagonists. Parallel experiments examining pain behavior will allow us to extend our findings past co-variance and to determine if blockade of any substance, within the DRG or spinal cord, is either necessary or sufficient to reduce pain behavior. Delineation of these patterns and mechanistic cause/effect relationships will increase our ability to methodologically develop rational treatments for neuropathic pain by providing us with more selective targets. This approach has the potential for avoiding some of the problems that occur with complete inhibition of p38 function.